Histone Deacetylase Inhibitors Target DNA Replication Regulators and Replication Stress in Ewing Sarcoma Cells

Histone deacetylases (HDAC) regulate diverse pathways in Cancer cells. Previously, we identified that Ewing sarcoma tumors, which are caused by a translocation between the EWSR1 and FLI1 genes (EWS::FLI1), are sensitive to drugs that target DNA replication, including the RRM1 and RRM2 subunits of ribonucleotide reductase, and the ATR-checkpoint kinase 1 (Chk1)-WEE1 signaling pathway. In this study, we identified that multiple HDAC inhibitors, including fimepinostat, romidepsin, and panobinostat, downregulate the levels of the RRM1, RRM2, Chk1, and Wee1 proteins in Ewing sarcoma cells and impair DNA replication. Moreover, transcriptome analyses identified that HDAC inhibitors downregulate the expression of multiple components of the prereplication complex, including the minichromosome maintenance complex 2-7 (MCM2-7) proteins and CDT1, that are essential for genomic DNA replication. Additionally, proteomic studies identified that HDAC inhibitors also downregulate the level of the BRD4 protein, a BET bromodomain protein that regulates both the transcriptional program of the EWS::FLI1 oncoprotein and DNA replication. Overall, these results provide novel insight into the molecular mechanisms by which HDAC inhibitors target Cancer cells, regulate DNA replication, and inhibit the cellular response to DNA replication stress.

Significance: In this work, we identify that HDAC inhibitors broadly disrupt DNA replication and the response to replication stress in Ewing sarcoma cells by downregulating RRM1, RRM2, Chk1, and Wee1. Notably, HDAC inhibitors also reduce MCM2-7 proteins, which are essential for prereplication complex helicase function in replication initiation and elongation, and CDT1, which loads MCM2-7 onto DNA.